This invention relates generally to fluorescence resonance energy transfer (FRET), and more particularly to calibration standards for FRET.
Fluorescence resonance energy transfer (FRET) is a non-radiative process whereby energy from a fluorescent donor molecule is transferred to an acceptor molecule without the involvement of a photon. Excitation of the donor molecule enhances the fluorescence emission of the longer-wavelength acceptor molecule (i.e., sensitized acceptor emission). The quantum yield of the donor fluorescence emission is concomitantly diminished. FRET has become a valuable tool for microscopy, because the efficiency of energy transfer has a strong inverse dependence on the distance between the donor and acceptor molecules. Thus, the appearance of FRET is a highly specific indicator of the proximity of the two molecules. This has led to the use of FRET efficiency as a xe2x80x9cspectroscopic rulerxe2x80x9d to measure molecular distances.
A typical FRET experiment for observing cells involves specifically labeling particular molecules with fluorescent dyes and detecting these dyes over selected excitation and emission wavelength ranges. These wavelength ranges are commonly referred to as xe2x80x9cchannelsxe2x80x9d for a particular fluorophore. Fluorescent labeling is easier to accomplish for extracellular molecules than for intracellular ones. For intracellular molecules, the dye conjugated molecules must be either injected into the cell for observation or the labeled molecules may be overlaid onto a thin section of fixed, permeabilized tissue. Recently, however, the availability of green fluorescent protein (GFP) mutants with shifted excitation and emission spectra has made it feasible to measure protein-protein interactions by using GFP tags as intracellular markers. GFP-tagged protein chimeras are expressed intracellularly and do not require any chemical treatment to become fluorescent. FRET can also occur between fusions of blue-emitting and green-emitting GFP variants.
Although FRET microscopy has the potential to become a widely used analytical tool in these systems, accurate steady-state measurement of FRET by standard epifluorescence microscopy has been hindered by several limitations, including:
(1) Bleed-through or spillover of donor (and isolated acceptor) fluorescence emission into the FRET channel due to spectral overlap. Compensation for spectral overlap requires the use of accurate calibration standards to determine the correction factors.
(2) Image registration. Correcting each pixel in an image for spectral overlap requires taking three separate images with each of the three epifluorescence cubes (i.e., donor emission, acceptor emission, and sensitized acceptor emission). If the surfaces of all three epifluorescence cubes are not exactly parallel, image registration is lost and the resultant geometric distortion will also have to be corrected. Loss of image registration greatly complicates the correction procedure and reduces the overall accuracy of the corrected data.
(3) Calculation of FRET by sensitized emission has a large uncertainty when the FRET efficiency is low.
(4) Photobleaching of the donor during the measurement results in anomalously low estimates of FRET in the sample.
These limitations apply to GFP as well as to other fluorescent FRET pairs.
Because of recent advances in xe2x80x9cchromosome painting,xe2x80x9d cell biologists are becoming familiar with epifluorescence microscopes that utilize multi-bandpass filters to obtain information on several dyes simultaneously. Some of these instruments employ interferometry, while others use filter wheels in the excitation path to select specific excitation bands. In both cases, an assumption is made that excitation at a given wavelength results in emission from the next reddest transmission band. While multiband cubes help to solve the image registration problem for separable dyes, fluorescence phenomena which involve large or variable Stokes shifts cannot be addressed. Multiband systems cannot be used to detect FRET because the FRET emission channel is not the next (redder) emission band and is therefore indistinguishable from the donor""s emission.
Accordingly, the inventors have determined that a need exists for an improved FRET system which provides for close image registration, and minimizes or corrects for low FRET efficiency, photobleaching, and spectral overlap. The present invention provides for such a system.
The invention provides a method for transforming an initial image of biological material to a processed image, the initial image being represented by a set of pixels each having three color space coordinates. The method includes the steps of (a) selecting at least two image regions in the initial image, each image region including at least one pixel, wherein the image regions define at least two distinguishable spectral categories; (b) performing a color space transformation on the initial image, based on the color space coordinates of the pixels in each of the selected image regions, to generate a processed image; and (c) evaluating the processed image for a visible indication of an improved spatial distribution of color.
The invention further provides a quantitative method for color space transforming an initial FRET image of biological material to a corrected FRET image, and includes applying other image corrections.
The invention also provides a set of calibration targets for FRET. The set of calibration targets includes: (a) a pure donor target for binding only to donor molecules; (b) a pure acceptor target for binding only to acceptor molecules; and (c) a donor and acceptor target for binding to a mixture of donor and acceptor molecules which exhibit FRET. In a specific embodiment, the set of calibration targets are genetically engineered derivatives of the Green Fluorescent Protein (GFP) bound to the surface of Ni chelating beads by histidine-tags (His-tags).